Broad host spectrum rhizobiaceae nodulation signals

ABSTRACT

Nod factors of general formula (I): ##STR1## wherein R 1 , R 2  and R 3  are hydrogen atom, a carbamyl group or an acetyl group; R 5  is the aliphatic chain of a fatty acid; n is 1-4; and one or more of substituents R 1 , R 2  and R 3  is a carbamyl group, and/or R 4  is a methyl group, and/or R 6  is an optionally substituted monosaccharide or oligosaccharide attached to the glucosamin via a glycoside bond.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the preparation of novel, broad host spectrumnodulation signals (Nod factors).

2. Description of the Related Art

Sol bacteria which belong to the genera Azorhizobium, Bradyrhizobium,Sinorhizobium and Rhizobium, (which are referred to under the generalterm rhizobia) are capable of interacting with the roots of legumes inorder to form nodules in which they fix atmospheric nitrogen. However,only certain combinations of bacteria and plants result in nodulationand host specificity of rhizobia varies greatly [LONG, Cell. 56, 203(1989)]; [MARTINEZ et al., Crit. Rev. Plant Sci., 23, 483 (1990)];[DENARIE et al., in Molecular Signals in Plant-Microbe Communications,D.P.S. Verma Ed. pp. 295-324 (CRC Press, Boca Raton, 1992)]. Certainrhizobia (for example R. leguminosarum and R. meliloti) form nodules ononly a small number of legume species, while, on the other hand, othershave a broader host spectrum and can form an association with a largenumber of plants.

Nodule formation results from a coordinated expression of plant genesand bacterial genes. The expression of rhizobial nodulation genes (nod)is controlled by nodD regulator genes whose products are activated byflavonoids which are secreted by the roots of the plants. The ability ofthe NodD proteins to interact with the plant flavonoids in a specificmanner defines a first level of host specificity.

Moreover, two categories of structural nod genes exist: genes which arein common and specific genes. The nodABC genes are common to allrhizobia, while nod genes, which are specific to the species, are themajor determinants of host specificity.

It has been shown that the common nod genes and the specific nod genesare simultaneously involved in the production of extracellular Nodfactors which cause deformation of root hairs in legumes. Some inventorshave identified Nod factors, termed NodRm, in R. meliloti which factorshave a lipo-oligosaccharide structure, whose biosynthesis is under thecontrol of common nodABC genes, and which are glucosamine oligomerslinked to each other by β-1,4 bonds, N-acylated on the non-reducingterminal glucosamine and N-acetylated on the other glucosamine residues(Application PCT FR/9100283 in the names of the INSTITUT NATIONAL DE LARECHERCHE AGRONOMIQUE and the CENTRE NATIONAL DE LA RECHERCHESCIENTIFIQUE). Host specificity is subsequently determined by the natureof the substituents attached to this skeleton which they have in common.In the case of R. meliloti, the function of major host specificity genes(nodH and nodPQ) is to determine the sulfation of theselipo-oligosaccharide factors [ROCHE et al., Cell, 67, 1131 (1991)],while, in the case of R. leguminosarum, the nodFE genes control thesynthesis of a highly unsaturated lipid residue [SPAINK et al., Nature,354, 125, (1991)].

The strain Rhizobium sp. NGR234 has a unique place amongst the legumesymbionts; it has, in fact, the broadest host spectrum of all knownrhizobia, and it is known at present that it causes nodulation of over60 legume species. Amongst these hosts there are, in particular, most ofthe commercially important legumes such as, for example, soya bean orgroundnut. Rhizobium NGR234 can, moreover, cause nodulation of plantswhich do not belong to the legumes, such as, for example, Parasponiaandersonii.

SUMMARY OF THE INVENTION

The inventors have sought to isolate and identify the Nod factors whichare responsible for the broad host spectrum of Rhizohium sp. NGR234 andwere able to characterize a novel family of Nod factors termed NodNGRfactors. These NodNGR factors are lipooligosaccharides which belong tothe same family as the NodRm factors which have already been describedby some of the inventors (Application PCT FR/9100283), but also havestructural characteristics which allow them to be distinguished from NodRm factors.

Firstly, their reducing terminal glucosamine residue is substituted onthe C6 by a different sugar;

Secondly, their non-reducing terminal glucosamine can be esterified byone or more carbamoyl groups;

Thirdly, the nitrogen atom which is substituted by the long-chain fattyacid is also methylated.

Moreover, the inventors have studied the structure of the Nod factorsproduced by a range of strains of the Rhizobiaceae from very differentgeographical origins and which are symbionts of a very wide range ofhosts, such as Rhizobium tropici which forms nodules on beans andLeucaena, Sinorhizobium fredii, which forms nodules on soya beans andAzorhizobium caulinodans which is a symbiont of Sesbania. They foundthat the Nod factors produced by these different strains had at leastone of the structural features observed for the NodNGR factors such asthe presence of a sugar on the reducing terminal glucosamine(Sinorhizobium fredii, Azorhizobium caulinodans, Rhizobium phaseoli) orof an N-methyl group on the non-reducing terminal glucosamine (Rhizobiumtropici, Azorhizobium caulinodans).

The novel Nod factors of the invention, which show at least one of thethree structural characteristics mentioned hereinabove, will generallybe termed hereinafter NodNGR-type Nod factors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the restriction map of the Eco RI fragment containing thenodD1 region from NGR234.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

More particularly, the NodNGR-type Nod factors obtained from NGR234 willhereinbelow be termed NodNGR factors. Nod factors obtained from otherstrains studied by the inventors and having at least one of thesecharacteristics represent NodNGR-type Nod factors. It seems that thesource of NodNGR-type Nod factors can be very varied.

The present invention relates to Nod factors of the general formula (I)hereinbelow: ##STR2## in which: R₁, R₂ and R₃ represent a hydrogen atom,a carbamoyl group or an acetyl group;

R₅ represents the aliphatic chain of a fatty acid;

n is between 1 and 4, and wherein:

one or more of the substituents R₁, R₂ or R₃ is a carbamoyl group,and/or

R₄ represents a methyl group, and/or

R₆ represents a monosaccharide or an oligosaccharide, optionallysubstituted, and linked to the glucosamine by a glycosidic linkage.

In a preferred embodiment of the present invention , R₅ represents thealiphatic chain of a C₁₀₋₂₄ -, preferably a C₁₄₋₂₀ -, fatty acid.

In a preferred form of this embodiment, R₅ is the aliphatic chain ofvaccenic acid or palmitic acid.

Vaccenic acid and palmitic acid are major substituents found inpreparations of NodNGR factors from Rhizobium NGR234; however, a largenumber of different fatty acids varying as much with regard to thenumber of carbon atoms of the aliphatic chain as by the degree ofunsaturation, or by the presence of substituents such as hydroxyl groupson said aliphatic chain, was observed; no effect of these variations onthe activity of the NodNGR factors was found by the inventors.

In yet another preferred embodiment of the present invention, R₆ isselected from the monosaccharide group comprising optionally substitutedfucose and optionally substituted arabinose.

In an advantageous arrangement of this embodiment, R₆ has the generalformula (II): ##STR3## in which: R₇ and R₈ represent a hydrogen atom, anacetyl group or a sulfate group,

R₁₀ represents a hydrogen atom or a methyl group.

In a preferred form of this arrangement, R₇ is a hydrogen atom and R₈ asulfate group.

In another preferred form of this arrangement, R₇ is an acetyl group andR₈ a hydrogen atom.

In yet another preferred form of this arrangement, both R₇ and R₈ arehydrogen atoms.

The NodNGR factors belong to the same family of molecules as the NodRmfactors which have been purified from R. meliloti: all have in commonthe skeleton of D-glucosamine residues linked to each other by β-1,4linkages, are N-acylated on the non-reducing terminal glucosamine andN-acetylated on the other residues. These structural similarities tie inwith the observations made before by some of the inventors and confirmthat, in all rhiozobia, the function of the nodABC genes is to controlthe synthesis of a skeletal structure of N-acylated and N-acetylatedoligochitosan. The work carried out by the inventors therefore revealsthat the Nod factors of all Rhizobiaceae belong to the same chemicalfamily.

In the NodRm factors, the fatty acid chain contains at least oneconjugated double bond which seems to play an essential role in theinduction of nodular meristems; in contrast, the NodNGR factors areN-acylated with vaccenic acid or palmitic acid and therefore do not havea conjugated double bond. The nitrogen atom which is substituted by along-chain fatty acid is also methylated in the NodNGR factors. Theseobservations allow the hypothesis to be put forward that certainbiological activities of the Nod-type lipo-oligosaccharides require acertain structural configuration at the joint between the lipid part andthe saccharide skeletal structure. These structural conditions would beprovided in one case by the conjugated double bond and, in the othercase by the N-methyl group.

The family of the NodNGR factors is very large. Mass spectrometryanalysis using fast-atom bombardment ionization (FAB-MS) and alsonuclear magnetic resonance analysis have shown that the followingvariations exist in the substituents:

1) The 2-O-methylfucose residue can be unsubstituted or else sulfated onO-3 or acetylated on O-4;

2) The nitrogen atom of the non-reducing terminal glucosamine can beacylated by palmitic acid or else by vaccenic acid;

3) The number of carbamoyl substituents on the non-reducing terminalglucosamine varies between zero and two.

The combinations of these possible different substituents lead to atleast 18 (=3×2×3) possible structures if the carbamoyl substitutionsites are fixed and their number may even be greater to the extent thatthe carbamoyl group substitution site, or sites, can vary betweenpositions O-3, O-4 and O-6. It is reasonable to assume that it is inparticular this structural diversity which is responsible for the broadhost spectrum of the NodNGR factors.

The invention also relates to rhizobia strains which overproduce NodNGRtype factors, which comprise at least one recombinant plasmid expressinga regulator gene nodD from NGR234 and in particular a rhizobium strainNGR234 which overproduces Nod factors, which strain is obtained byintroducing, into NGR234, a recombinant multicopy plasmid termed pA28which expresses a regulator gene nodD from NGR234, so as to increase thenumber of copies of this gene, which results in an at least 10-foldincrease of the amount of Nod factors produced.

The invention also encompasses recombinant plasmids carrying theregulator gene nodD1 from NGR234, in particular plasmid pA28, whichresults from inserting an EcoRI-PstI fragment from plasmid pNGRH6[BASSAM et al. Mol. Plant-Microbe Interact, 1, 161, (1988)], whichcarries the nodD1 region from NGR234, into plasmid pRK7813 [JONES andGUTTERSON, Gene, 61, 299-306, (1987)].

The invention also relates to a process for the preparation of NodNGRfactors, or NodNGR-type factors, which process comprises a step in whichat least one strain of rhizobia producing said Nod factors is cultured.

Preferably, a strain will be chosen into which a plasmid according tothe invention has been introduced.

In a preferred embodiment of the preparation process of NodNGR-typefactors according to the invention, it comprises, moreover, a step inwhich one or more fractions comprising said factors are extracted fromsaid culture of rhizobia strains.

In a preferred arrangement of this embodiment, the NodNGR factors areextracted from culture supernatant by reverse-phase chromatography, byabsorption on a silica column to which hydrophobic groups, such asoctadecyl residues, are grafted, followed by elution with methanol.

The present invention also relates to a plant treatment agentcomprising, as active ingredient, at least one NodNGR factor orNodNGR-type factor as defined further above, which can be used inparticular:

as an agent for stimulating symbiotic properties of legumes, especiallywith regard to nitrogen fixation;

as an agent for stimulating the defence mechanisms of plants againstpathogene.

Said plant treatment agent preferably comprises a mixture of NodNGRfactors and/or NodNGR-type factors. It can advantageously also compriseother Nod factors, for example NodRm-type factors.

In an advantageous embodiment of the plant treatment agent according tothe present invention, the composition is included in a solid carrier,such as granules, or else formulated in the form of a coatingcomposition for seed or an aqueous solution or suspension for spraying,in which a Nod factor or Nod factors, according to the invention arepresent alone or in association with other components, such as, forexample, other Nod factors.

In another advantageous embodiment of the plant treatment agentaccording to the present invention, a Nod factor, or each of theingredients of a mixture of Nod factors, according to the invention arepresent in the coating compositions or in the aqueous solutions orsuspensions at a concentration of between 10⁻⁶ M and 10⁻¹⁴ M.

Moreover, the present invention relates to a therapeutic agentcomprising, as active ingredient, at least one NodNGR factor orNodNGR-type factor as defined further above.

In an advantageous embodiment of this therapeutic agent, said factor ispresent in the therapeutic agent at a concentration of between 10⁻⁴ Mand 10⁻⁸ M.

Besides the above arrangements, the invention also encompasses otherarrangements which will emerge from the description which follows.

It must be understood, however, that these examples as well as theappended drawings are given only by way of illustrating the subject ofthe invention but without imposing any limitation whatsoever.

EXAMPLE 1

PRODUCTION OF A STRAIN OF RHIZOBIACEAE BACTERIA WHICH OVERPRODUCE NodNGRFACTORS

Strain NGR234 and its DNA were engineered as described by BROUGHTON etal. (Arch. Microbial. 141, 14 (1985)) and PERRET et al. (Proc. Natl.Acad. Sci. 88, 1923 (1991)), or by means of traditional techniques (J.SAMBROOK et al., Molecular Cloning. A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor (1989)).

A 2.9 kb EcoR1 fragment containing the nodD1 region from NRG234 wasexcised from plasmid pNGRH6 [BASSAM et al., Mol., Plant-MicrobeInteract. 1, 161, (1988)] and then digested with Pst1. The resulting 2.2kb EcoR1-Pst1 fragment was cloned into pRK7813 at the Pst1 site to giveplasmid pA28. pA28 is reintroduced into Rhizobium sp. NGR234 (Rif^(R))by means of conjugation. The resulting strain NGR(pA28) overproduces Nodfactors. It is Nod⁺ on L. leucocephala, M. atropurpureum, and V.unguiculata. FIG. 1 shows the restriction map of the EcoR1 fragment. Therestriction sites are designated as follows:

    ______________________________________                                        B = BamH1,     C = Cla1,    E = EcoR1,                                        P = Psi1,      S = Sst1.                                                      ______________________________________                                    

EXAMPLE 2

PURIFICATION OF THE NodNGR FACTORS

The bacteria NGR234 or NGR234(pa28) are cultured at 27° C. on B+D medium(W. J. BROUGHTON and M. J. DILWORTH, Biochem. J. 125, 1075 (1971))containing 12 mM succinate, 6 mM glutamate, 1 ml/litre of GAMBORG'sVitamin B5 solution (Sigma, St, Louis, Mo.) (=RMM3) to the end of thelogarithmic phase.

For analysis by thin-layer chromatography, 30 ml of cultured bacteria(NGR 234) are induced using 10⁻⁶ M of apigenin, which induces theexpression of the nodD genes from NGR234, in the presence of 1 μCi/ml ofsodium sulfate labeled with sulfur-35, or sodium acetate labeled withcarbon-14.

The supernatants are extracted on SEPPAK C₁₈ cartridges (Waters Assoc.,Milford, Mass.), washed with distilled water and eluted using methanol.The concentrated methanol extracts are applied to silica gel 60 G plates(MERCK, DARMSTADT); chromatography is carried out in a chloroform:methanol: 5M NH₄ OH (5:4:1 by volume) mixture, and the plate is placedon a FUJI RX film (Fuji Photo Film Co, TOKYO).

Induction of nod genes by 10⁻⁶ M apigenin in the presence of ¹⁴C-labeled acetate or ³⁵ S-labeled sulfate results in two radioactivespots on the TLC plates. Extraction of the active principles containedin each of these spots allows substances to be obtained which causedeformation of the root hairs of Macroptilium. This demonstrates thatNGR234 secretes sulfur-containing Nod factors. On the other hand, nospot obtained from an NGR234 derivative can be detected in which thenodABC genes have been deleted. Moreover, if the number of copies of thenodD1 gene (regulator gene) is increased by introducing plasmid pA28into NGR234, an increased production of Nod factors is observed, theMount produced being multiplied by a factor of 5 to 10.

For chemical analysis, the Nod factors were isolated from the culturesupernatant of NGR234 containing plasmid pA28, following induction withapigenin. This production on a larger scale is effected using 50 litresof NGR234(pa28) bacterial culture which has previously been induced withapigenin in RMM3 medium. The lipophilic material is recovered on a C₁₈reversed-phase column (LICHROSORB-18, 40 μ, MERCK, DARMSTADT). Thecolumn is washed with 50 times its volume of distilled water and elutedwith 10 volumes of methanol. The methanolic solution is evaporated invacuo and diluted with 100 ml of distilled water. After filtration, theaqueous solution is extracted using 50 ml of ethyl acetate to extract,in particular, apigenin. The aqueous solution is concentrated, and thewater-soluble constituents are separated by preparative HPLC on a C₁₈reversed-phase column. Elution is monitored at 206 nm. The solvent is agradient of acetonitrile in water.

Two major peaks termed fractions A and B were collected in this way.Fraction A (0.3 mg/l of the starting culture) co-elutes with thematerial originating from the culture labeled with sulfur-35, whilefraction B (0.5 mg/l of the original culture) is not labeled under theseconditions. The comparison of biological activities of these twofractions is described hereinbelow in Example 4.

EXAMPLE 3

CHARACTERIZATION OF THE NodNGR FACTORS

Hydrolysis with trifluoroacetic acid (4M for 4 hours at 100° C.) of eachof the two fractions liberates sugars and fatty acids. The sugars wereidentified as D-glucosamine, N-methyl-D-glucosamine and2-O-methyl-L-fucose, either by gas chromatography, mass spectrometry,(GC-MS) of their alditol acetate derivatives, or else by gaschromatographic analysis of their (+)-2-butanolglycosides. Two acidswere identified: the largest component as being vaccenic acid(11-Z-octadecenoic acid), while the minor component (approximately 20%of the total) was identified as palmitic acid. The existence of askeleton which they have in common and which is composed of pentamericN-acetyl-glucosamine oligomers having a plurality of substituents wasdeduced from the FAB mass spectrum, which reveals series of ionsseparated by 203 mass units (molar mass of an N-acetylglucosamineresidue).

Fraction A is a mixture of a plurality of sulfated compounds, asconfirmed by the ease with which the SO₃ radical is lost in positiveionized form. The molecular weight of the major component, deduced fromthe spectrum of negative ions, is 1595. Other components with a mass ofless than 43 or 26 mass units, or a combination of the two, weredetected. This latter difference corresponds to the difference betweenthe molecular weight of vaccenic acid and the molecular weight ofpalmitic acid. Equally, the difference of 43 mass units, which wasrepeated twice, was attributed to the presence or absence of additionalCO--NH groups (carbamoyl residues). The fact that this pattern of threepeaks which are separated by 43 mass units accompanied by satellitepeaks at a distance of 26 mass units is observed each time a glycosidiclinkage is ruptured in the form of positive ions (formation of oxeniumions) justifies the localization of carbamoyl groups of the non-reducingterminal glucosamine. Moreover, if the mass of the oxenium ions of m/z440, 483 and 526 is subtracted from the mass of a vaccenyl residue(ketene) and, if appropriate, the mass of zero, one or two carbamoylgroups (43 mass units), the mass of the oxenium ion of amethylglucosamine is obtained. This allows the N-methylglucosamine to belocalized at the non-reducing end of the oligosaccharide.

Fraction B is also a mixture. Two major components were identified(molecular weights 1557 and 1515, respectively). The difference of 42mass units between these two components suggests that the second is amonoacetylated form of the first. On the other hand, as in fraction A,other components in which the mass is lower than 43 or 26 mass units arealso present. As in the case of the components of fraction A, thecarbamoyl groups are localized on the non-reducing terminalN-methylglucosamine which has carries the N-acyl group. In contrast, theadditional acetyl group, which is not present in any of the oxenium ionsobserved, is localized near the reducing end.

The carbon-13 NMR spectrum is compatible with the presence of carbamoylgroups (δ=161.09, 160.62 and 159.80 ppm) and the presence of the othersubstituents described above. The proton NMR spectrum attributes βconfigurations to the linkages between glucosamines and α configurationsfor the linkage between 2-O-methylfucose and the reducing glucosamine.The COSY spectrum shows a correlation between the H-5 of the fucose anda deshielded proton δ=4.53 ppm in the case of the compounds of fractionB. This allows the position of the acetyl group to be attributed to the0-4 position of 2-O-methylfucose. In parallel, the COSY spectrum of thecompound of fraction A shows a correlation between the H-2 of2-O-methylfucose (3.67 ppm) and the deshielded H-3 proton at δ=4.65 ppm,which allows the sulfate group to be localized at the O-3 of2-O-methylfucose. This latter attribution is confirmed by analysis ofthe sugars obtained by hydrolysis of the reduced and permethylatedfraction A, which shows the presence of dimethyl-2,4-fucose. Moreover,by identifying trimethyl-1,2,3,5-glucosaminitol in the hydrolysisproducts of the reduced and permethylated fractions A and B, it can beconfirmed that the methylfucose is linked glycosidically to the O-6 ofthe reducing glucosamine. Finally, this analysis also shows 1-4 linkagesbetween the various glucosamines. Since the methylation conditionsresult in the simultaneous elimination the ester groups (acetates andcarbamates), the position of these groups cannot be determined by thismethod. However, the major components of fractions A and B arebicarbamylated while the types which lack carbamoyl substituents are inthe minority.

EXAMPLE 4

TEST FOR GROWTH OF ROOT HAIRS (Hai) AND DEFORMATION OF ROOT HAIRS (Had)CAUSED BY NOD-SULFATED AND NON-SULFATED FACTORS FROM RHIZOBIUM NGR234 ONMACROPTILIUM ATROPURPUREUM, MEDICAGO SATIVA, VICIA SATIVA AND VIGNAUNGUICULATA

The two fractions A and B, (sulfated and non-sulfated) were separated bya reverse-phase HPLC chromatography following the protocol described inExample 2 and were tested separately for their biological activity.

The Had test (deformation of root hairs) on M. sativa and Hai test(proliferation and bending of the root hairs) on V. sativa were carriedout as described by ROCHE et al. [Cell., 76, 1131 (1991)]. In the Badtests on Macroptilium and Vigna, sterile plantlets are placed intomodified Eppendorf tubes (with the cap and part of the bottom removed),and the Eppendorf tubes containing the plantlets are suspended into testtubes whose bottom is painted black in order to protect the roots fromlight, in such a manner that the root tip is in contact with 10 ml ofB+D medium. After incubation for 60 hours (16-hour day, 30° C.; 8-hournight, 20° C.), the roots are removed, stained with Methylene Blue andexamined under an inverted microscope. Those root systems which clearlyshow branching or bending (prolific ramifications or bending at morethan one point in the root system) are termed Had⁺. Those roots whichare covered in root hairs are termed Hai⁺. 10 plants were used for eachtreatment and dilution. Moreover, 40 (Macroptilium and Vigna) and 60(Medicago and Vicia) plantlets are used as control plants (grown onmedium only) in order to estimate the intrinsic variability of thecharacters Bad and Hai between one plant and another.

The results are shown in Table I hereinbelow. These results show thenumber of plants (above 10) which show a positive Bad or Hai activity.The numbers are followed by ^(S) if the ratio of Had⁺ or Hai⁺ issignificantly higher (probability P=0.05) in the treated plants than inthe controls (analyzed using the Fisher test).

NodRm-IV (Ac,S) is the major sulfated Nod factor of strain R. meliloti2011. It is used as positive control for Bad activity on Medicago.

NodRm-IV(Ac) is a non-sulfated Nod factor of NodH⁻ mutants of R.meliloti. It is used as positive control for Hai activity on Vicia.

                                      TABLE 1                                     __________________________________________________________________________    CONCENTRATION Nod FACTOR (M)                                                            10.sup.-7                                                                         10.sup.-8                                                                        10.sup.-9                                                                         10.sup.-10                                                                         10.sup.-11                                                                        10.sup.-12                                                                         10.sup.-13                                 __________________________________________________________________________    Macroptilium                                                                  (Had)                                                                         NodNRG    nt  10.sup.s                                                                         10.sup.s                                                                          7.sup.s                                                                            3.sup.s                                                                           0                                               sulfated                                                                      NodNGR    nt  10.sup.s                                                                         9.sup.s                                                                           4.sup.s                                                                            1   0                                               non-sulfated                                                                  Vigna                                                                         (Had)                                                                         NodNRG    10.sup.s                                                                          10.sup.s                                                                         10.sup.s                                                                          10.sup.s                                                                           10.sup.s                                                                          9.sup.s                                                                            6.sup.s                                    sulfated                                                                      NodNGR    10.sup.s                                                                          10.sup.s                                                                         10.sup.s                                                                          8.sup.s                                                                            5.sup.s                                                                           4    nt                                         non-sulfated                                                                  Medicago                                                                      (Had)                                                                         NodNRG    9.sup.s                                                                           8.sup.s                                                                          9.sup.s                                                                           5.sup.s                                                                            3.sup.s                                                                           1                                               sulfated                                                                      NodNGR    5.sup.s                                                                           1  2   0    1   1                                               non-sulfated                                                                  NodRM-IV  nt  9.sup.s                                                                          8.sup.s                                                                           7.sup.s                                                                            6.sup.s                                                                           4.sup.s                                         (AC,S)                                                                        Vicia                                                                         (Hai)                                                                         NodNRG    4.sup.s                                                                           5.sup.s                                                                          3.sup.s                                                                           0    0   0                                               sulfated                                                                      NodNGR    10.sup.s                                                                          10.sup.s                                                                         9.sup.s                                                                           2.sup.s                                                                            0   0                                               non-sulfated                                                                  NodRM-IV (Ac)                                                                           nt  10.sup.s                                                                         10.sup.s                                                                          8.sup.s                                                                            2.sup.s                                                                           0                                               __________________________________________________________________________     nt = non tested                                                          

In root hair deformation tests (Had) carried out with host plants ofNGR234, the two groups of NodNGR factors are active at concentrations ofas little as 10⁻¹⁰ M/10⁻¹¹ M in Macroptilium and 10⁻¹¹ M/10⁻¹² M inVigna. Moreover, in Vigna, the NodRm factors induce not onlydeformations of the hairiness of the roots, but also of the appearanceof a large number of root hairs, as well as bending of the root hairs(Hai).

The sulfated NodRm factors obtained from R. meliloti are Had⁺ inMedicago sativa, and Hai⁻ in Vicia sativa supsp. nigra. In contrast, thenon-sulfated NodRm factors secreted by NodH⁻ mutants of R. meliloti areHad⁻ in Medicago and Hai⁺ in Vicia. Interestingly, sulfated andnon-sulfated NodNGR factors have a biological activity on both legumes.In Medicago, the sulfated NodNGR factors are 10,000 times more activethan the non-sulfated factors and cause deformation of the hairiness ofthe roots at concentrations of less than 10⁻¹¹ mole. The sulfated NodNGRfactors differ from the sulfated NodRm factors with regard to a largenumber of criteria: for example, the presence of carbamoyl groups and ofa methylfucose residue, localization of the sulfate group on the fucoseinstead of the glucosamine, the absence of a conjugated double bond onthe acyl chain which substitutes the nitrogen, and the presence of amethyl group which substitutes the nitrogen. However, both types offactors are active in Medicago, and their activity decreases by a factorof approximately 10,000 when the sulfate group is removed, whichdemonstrates that Medicago is highly sensitive to sulfated Nod factors.In contrast, in Vicia, the non-sulfated compounds are more active, anddeformation of the hairiness of the roots is observed at concentrationsof less than 10⁻¹¹ M.

We claim:
 1. A NodNGR-type factor of the following formula (I): ##STR4##in which: R₁, R₂ and R₃ represent a hydrogen atom, a carbamoyl group oran acetyl group;R₅ represents the aliphatic chain of a fatty acid; n isbetween 1 and 4, and wherein:one or more of the substituents R₁, R₂ orR₃ is a carbamoyl group, and/or R₄ represents a methyl group, and/or R₆has the general formula (II) ##STR5## in which: R₇ and R₈ represent ahydrogen atom, an acetyl group or a sulfate group, R₁₀ represents ahydrogen atom or a methyl group.
 2. The Nod factor as claimed in claim1, wherein R₅ represents the aliphatic chain of a C₁₀₋₂₄ fatty acid. 3.The Nod factor as claimed in claim 1, wherein R₇ is a hydrogen atom andR₈ is a sulfate group.
 4. The Nod factor as claimed in claim 1, whereinR₇ is an acetyl group and R₈ is a hydrogen atom.
 5. The Nod factor asclaimed in claim 1, wherein both R₇ and R₈ are hydrogen atoms.
 6. Aprocess for the preparation of NodNGR-type factors as claimed in claim1, which process comprises a step in which at least one strain ofRhizobiaceae producing said Nod factors in cultured, and a step in whichsaid NodNGR factors are purified from the culture.
 7. Plant treatmentagent, which comprises, as active ingredient, at least one NodNGR-typefactor or a mixture of NodNGR-type factors as claimed in claim
 2. 8. TheNod factor as claimed in claim 2, wherein R₅ is the aliphatic chain ofvaccenic acid or palmitic acid.
 9. The Nod factor as claimed in claim 2,wherein R₅ represents the aliphatic chain of a C₁₄₋₂₀ fatty acid. 10.The process as claimed in claim 6, wherein said strain is a strain whichoverproduces Nod NGR factors and comprises at least one plasmid encodingnodD1 from Rhizobium, Azorhizobium, Bradyrhizobium or Sinorhizobium. 11.The plant treatment agent as claimed in claim 7, which comprises,additionally, other Nod factors.
 12. The plant treatment agent asclaimed in claim 7, which is included in a solid carrier or formulatedin the form of a coating composition for seed or an aqueous solution orsuspension for spraying.
 13. The plant treatment agent as claimed inclaim 7, wherein the NODNGR-type factor or each of the ingredients ofthe mixture of NodNGR-type factors is present at a concentration ofbetween 10⁻⁶ M and 10⁻¹⁴ M.
 14. The process as claimed in claim 10,wherein the plasmid results from inserting an Eco I-Pst I fragment ofplasmid pNGRH6 comprising the regulator gene nodD1 from strain NGR234into plasmid pRK7813.
 15. A strain of Rhizobiaceae which overproducesNodNGR-type factors for carrying out the process as claimed in claim 10,which comprises at least one recombinant plasmid which results frominserting an EcoRI-PstI fragment of plasmid pNGRH6 comprising aregulator gene nodD1 from strain NGR234 into plasmid pRK7813.
 16. Theprocess as claimed in ether of claims 6 or 14, which comprises,moreover, a step in which one or more fractions comprising theNodNGR-type factors are extracted from said culture of Rhizobiaceae. 17.The process as claimed in claim 16, wherein the NodNGR-type factors areextracted from culture supernatant by absorption onto a silica column towhich hydrophobic groups are grafted, following by elution withmethanol.